The organization of a cell is designed for optimal structure and function. Each molecule produced by the cell must be targeted to the appropriate subcellular location. To be properly localized, each molecule must contain information which provides its cellular address. Trafficking machinery can use this information to appropriately transport the molecule to its destination. This is a dynamic process, and the location of a molecule can change over time. Defects in any step involving a molecule's proper localization can result in a disorder, such as diabetes or Alzheimer's disease. (James, D. E., and Piper, R. C. (1994) J. Cell Biol. 126:1123-1126; and Nordstedt, C., et al. (1993) J. Biol. Chem. 268:608-612.)
The information that provides the address for targeting resides within the primary structure of the protein. Certain amino acid sequences act as “delivery codes” during the processing or recycling of a molecule and assure that the molecule is properly localized. Several motifs have been identified. The two principal motifs are the nuclear localization signal and signal peptide. Nuclear localization signals (NLS) consist of short stretches of amino acids enriched in basic residues. NLS are found on proteins that are targeted to the nucleus, such as the glucocorticoid receptor. The NLS is recognized by the NLS receptor, importin, which then interacts with the monomeric GTP-binding protein Ran. This NLS protein/receptor/Ran complex navigates the nuclear pore with the help of the homodimeric protein nuclear transport factor 2 (NTF2). NTF2 binds to the GDP-bound form of Ran and to multiple proteins of the nuclear pore complex, such as p62.
Signal peptides are found on proteins that are targeted to the endoplasmic reticulum (ER). Signal peptides consist of stretches of amino acids enriched in hydrophobic residues. Signal peptides are usually found at the extreme N-terminus of the protein and are recognized by a cytosolic signal-recognition peptide (SRP). The SRP binds to the signal peptide and to an SRP receptor, an integral membrane protein in the ER. Once bound to the SRP receptor, the newly formed protein containing the signal peptide is translocated across the ER membrane. Proteins containing signal peptides may end up inserted into the lipid bilayer, or they may end up in the lumen of an organelle or secreted from the cell.
Proteins may also contain separate motifs that specify delivery or retention in a subcellular location. For example, the trans-Golgi network integral membrane protein TGN38 cycles between the trans-Golgi network (TGN) and the plasma membrane. TGN38 contains two separate motifs. One motif, located within the cytoplasmic tail, is responsible for delivery to the Golgi. A second motif, located within the hydrophobic membrane-spanning region, is responsible for retention of the protein in the TGN. (Stephens, D. J., et al. (1997) J. Biol. Chem. 272:14104-14109.) Modification of the motif may alter the address for a protein and cause it to be relocalized. For example, plasma membrane receptors, such as the epidermal growth factor (EGF) receptor and the T-cell receptor (TCR), contain targeting motifs which, when ligand binds to the receptor, become phosphorylated. Phosphorylation of the targeting motif results in internalization and delivery of the receptor to the lysosome for degradation. (Dietrich, J. et al. (1994) EMBO J. 13:2156-2166.)
The information provided by the amino acid motifs is used by the trafficking machinery to sequester and package the protein into vesicles, and then deliver it to the appropriate location. Sorting nexin-1 (SNX1) is an example of a protein involved in the recognition of motifs involved in lysosomal targeting. Molecules targeted for the lysosome that require SNX1 include the carboxypeptidase Y sorting receptor and the EGF receptor. In the absence of SNX1, these molecules become mislocalized. (Kurten, R. C., Cadena, D. L., and Gill, G. N. (1996) Science 272:1008-1010; and Horazdovsky, B. F. et al. (1997) Mol. Biol. Cell 8:1529-1541.) The adaptor protein (AP) complex, which triggers assembly of clathrin on membranes to form vesicles, is also involved in sequestration of proteins for delivery to subcellular locations. AP-1 vesicles, which form at the Golgi, include cation-independent and cation-dependent mannose-6-phosphate receptors (MPRs). These vesicles are delivered to the lysosome. AP-2 vesicles, which form at the plasma membrane, include TGN38 (Stephens, supra) and ligand-bound TCR (Dietrich, supra.) TGN38-containing vesicles are delivered to the TGN and TCR-containing vesicles are delivered to the lysosome.
Once molecules containing targeting motifs have been sequestered and packaged into vesicles, the vesicles need to be delivered to the appropriate location. This delivery process involves another set of proteins. The Rab family of GTPases are involved in this process via a mechanism not clearly understood. Several Rab proteins have been described, each associated with a particular organelle. For example, Rab3 is associated with the plasma membrane, Rab4 with the sorting endosome, and Rab11 with the recycling endosome. There are also Rabs specific to cell types and functions. For example, Rab3A is specifically involved in synaptic vesicle exocytosis in stimulated nerve cells, and Rab4b is involved in glucose transporter-4 (GluT4) translocation in insulin-stimulated adipocytes. (James and Piper, supra.)
The discovery of new protein transport associated molecules and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cell proliferative and secretory disorders.